1. Field of the Invention
The present invention is directed generally to treating organisms, particularly aquatic organisms susceptible to a virus, such as an aquatic birnavirus infection, by administering compounds that increase the survival the subjects when exposed to or infected with an virus. The invention is directed to a prophylactic/therapeutic method for increasing the survival of aquatic species, particularly fish populations, that may be or are infected with a virus such as an aquatic birnavirus, and more particularly to a composition for the prevention and treatment of infectious pancreatic necrosis virus (IPNV) in fish comprising isoxazol compounds and/or aminoglycoside compounds. The invention is also directed to a prophylactic/therapeutic method for increasing the survival of animal species, particularly poultry and pig populations, that may be or are infected with a virus such as a birnavirus, ortomixovirus, picornavirus and to a composition for the prevention and treatment of infectious bursal disease virus (IBDV) in poultry comprising isoxazol compounds and/or aminoglycoside compounds.
2. Background
It is estimated that fish diseases cost twenty to thirty cents for each dollar spent rearing fish in the United States. Although fish pathogens include fungal, protozoan, and bacterial agents, it is the viral diseases that most concern fish farmers, hatchery managers, and scientists because they are largely uncontrollable. Fish are susceptible to a variety of viral infections and diseases. Such viruses include IPNV, pillar cell necrosis virus (PCNV), infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus (VHSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), shrimp white spot virus (WSV), Taura syndrome virus (TSV), hepatopancreatic parvovirus (HPV), infectious salmon anemia virus (ISAV), as well as others in the following families: Birnaviridae, Rhabdoviridae, Iridoviridae, Reoviridae, Ortomixovirus, and Picornavirus. For example, aquatic birnaviruses infect over 63 marine and fresh water organisms such as fish, shrimp and other crustaceans, oysters and other mollusks. The aquatic birnavirus, IPNV, is the causative agent of pancreatic disease in fish. Other birnaviridae species are known, such as IBDV, tellina virus and oyster virus.
IPNV and IBDV are members of the Birnaviridae family. They are an unenveloped icosahedric virus of approximately 60 nm in diameter with a genome consisting of two segments of double-stranded RNA.
Infectious pancreatic necrosis virus (IPNV) is a contagious viral disease in a variety of aquatic animals. In fish, IPNV causes morbidity and mortality in rainbow trout, Atlantic salmon, Pacific salmon, brook trout and other salmonids, especially fry, smolt and juvenile stages. IPNV is capable of infecting a number of different hosts and has a worldwide presence. Pilcher et al., Crit. Rev. Microbiol. 7:287 (1980), the contents of which are incorporated by reference in its entirety. IPNV has been isolated in a variety of aquatic animal species throughout the world, including various trout and salmon species, carp, perch, pike, eels, char, mollusks and crustaceans. Examples of species of fish and crustaceans/shellfish from which IPNV and viruses similar to IPNV have been isolated is found in Table 1. For example, IPNV has been found in such aquatic animals as Atlantic Salmon, farmed rainbow trout Oncorhynchus mykiss, wild flounder Rhombosolea tapirina, cod Pseudophycis sp., spiked dogfish Squalus megalops and ling Genypterus blacodes. Crane et al., Dis. Aquat. Organ. 43:1 (2000). IPNV has been isolated from salmonid and non-salmonid species of fish that lack pathogenic symptoms.
Infectious bursal disease virus (IBDV) is also a contagious viral disease in a variety of birds. IBDV affects the bursa of Fabricius, an organ that produces lymphocytes that help protect birds against disease and illness. IBDV attacks the bursa, killing lymphoid cells and suppressing the birds' immune systems. Clinical symptoms of IBDV include diarrhea, weight depression, paleness, and lameness. These symptoms result in increased condemnations of bird carcasses, above average mortality, and poor feed conversion and weight gain. IBDV causes serious problems for commercial poultry producers throughout the world, costing millions of dollars annually in production losses and treatment costs, and amounting to over a hundred million dollars in losses each year worldwide. IBDV is able to rapidly produce mutated viruses, called variant strains, that are resistant to vaccines.
TABLE 1Species of Fish and Crustaceans / ShellfishFrom Which IPNV and Viruses Similar to IPNV Have Been IsolatedScientific NameCommon Name (English or Spanish)SalmonHucho huchoDanube SalmonOncorhynchus gorbuschaPink SalmonOncorhynchus ketaChum SalmonOncorhynchus kisutchCoho SalmonOncorhynchus masouCherr SalmonOncorhynchus mykissRainbow TroutOncorhynchus nerkaSockeye SalmonOncorhynchus rhodurusAmago salmonOncorhynchus tshawytschaChinook SalmonSalmo clarkiCutthroat troutSalmo gairdneriRainbow troutSalmo salarAtlantic SalmonSalvelinus alpinusArctic charrSalvelinus namaycushLake troutSalvelinus pluviusJapanese chare, white spottedThymalus thymalusGraylingAdditional FishAbramis braxaBreamAlosa aestivalisBlueback herringAlosa sapidissimaAmerican shadAnguilla anguillaEuropean eelAnguilla japonicaJapanese eelBarbus barbusBarbelBlicca djoerknaSilver breamBrachydanio rerioZebra danioBrevoortia tyrannusAtlantic menhadenCarassius auratusGoldfishCarassius carassiusCrucion carpCatostomus commersoniWhite suckerChondrostoma nasusSheapCobitis sp.Spined bachCyprinus carpioCarpDicentrarchus labraxBass (sea bass)Esox luciusNorthern pikeEsox nigerChain pickerelLampetra fluviatilusRiver LampreyLeistomus xanthurusSpotLeuciscus rutilusRoachMenidia menidiaAtlantic SilversideMorone saxatilisStriped BassOxyeleotris matmoratusMarble GlobyParalichthys lethostigmaSouthern flounderPerca fluviatilisRedfin PerchPeudorasbora parvaGobioPhoxinus phoxinusMinnowScardinius arythophthalmusLeuciscoScophtalmus maximusTurbotSeriola quinquerodiataJapanese Amber JackSolea soleaCommon soleStizostedion vitreum vitreumPungent ThroatSymphysodon discusTilapia mossambicaAucunTrinectes maculatusShellfish / CrustaceansCarcinus masnasShore CrabCrassostrea gigasJapanese oysterCrassostrea virginicaEastern oysterLittorina littoreaCommon periwinkleMercenaria mercenariaHard clamMytilus edulisCommon musselOstrea edulisNative oysterPatella vulgataCommon limpetPenasus japonicusTellina tenuisThin tellis
In survivors of an IPNV epizootic, the virus persists and can cause severe growth retardation in the individual fish that exhibit virus persistence. McKnight et al., Br. Vet. J. 132: 76 (1976). In smolts, IPNV produces considerable necrosis or inflammation of the pancreas. Acute disease has been reported primarily in a limited number of salmonid species, such as trout and salmon. Many of these species are of great economic importance. Because mortality can be as high as 90 percent, an IPNV outbreak in a hatchery can be an economic disaster. Pilcher et al., Crit. Rev. Microbiol. 7:287 (1980).
The most susceptible age for IPNV infection in fish is in young fish, especially those that are two- to four-month old, in which such infections result in high mortality. Wolf et al., U.S. Dept. Int. Bur. Sport Fish and Wildlife Fish Disease Leaflet 1:14 (1966); Frantsi et al., J. Wildlife Dis. 7:249 (1971). In trout, IPNV usually attacks young fry about five to six weeks after their first feeding. IPNV is known to affect fish in their first year in salt water and to spread rapidly in farmed fish held in sea cages. Affected fish are thin, anorexic and lethargic with a tendency to congregate in cage corners and to fail to maintain a horizontal position. Ferguson et al., J. Fish Dis. 9:95 (1986). The affected fish are darker than usual, have slightly bulging eyes and often have swollen bellies. At the beginning of an outbreak, large numbers of slow, dark fry are seen up against water outflows, and fish are seen “shivering” near the surface. The shivering results from a characteristic symptom of the disease, a violent whirling form of swimming in which the fish rotate about their long axis. If the internal anatomy of the affected fish is examined, a characteristic white mucus is seen in the stomach. The pancreas appears to be the primary target organ for the virus. McKnight et al., Br. Vet. J. 132:76 (1976).
After an IPNV outbreak, the surviving fish generally become carriers of the virus. Fish that carry the virus are a serious problem for the aquaculture industry because the only control method currently available for eliminating the virus in carrier fish is complete destruction of these fish. Several factors appear to influence the severity of infection and the subsequent establishment of the carrier state. These factors include age, species, and water temperature. Surviving carriers shed infectious IPNV for the remainder of their lifetime, which is detectable in their fecal matter and sex products. Billi et al., J. Fish. Res. Bd. Can. 26:1459 (1969); Yamamoto, Can. J. Micro. 21:1343 (1975); and Reno et al., J. Fish. Res. Bd. Can. 33:1451 (1978).
The persistence of the virus in carrier fish appears to be the result of continued virus production by a small number of infected cells in certain organs. Hedrick, Ph.D. Thesis, “Persistent Infections of Salmonid Cell Lines with Infectious Pancreatic Necrosis Virus: A Model for the Carrier State in Trout,” Oregon State University, 1980. For IPNV, there are at least 9 type strains of Serogroup A and 4 other representative strains of Serotype A1. IPNV Serotype A1 is the predominant aquatic birnavirus and IPNV serotype in the United States.The family Birnaviridae describes and classifies a group of viruses, birnaviruses, which carry a bisegmented double-stranded RNA genome as their prominent characteristic; the two segments are called segment A and B. The RNA is enclosed in a non-enveloped icosahedral capsid, about 60 nm in diameter, which is arranged as a single shell. The two main representatives of this virus family are the infectious pancreatic necrosis virus of fish (IPNV) and the causative agent of infectious bursal disease of chickens (IBDV). The RNA of both IPNV and IBDV is covalently linked to a high molecular weight polypeptide, which is approximately 100 kDa. Birnaviruses have at least four structural proteins: named VP1, VP2, VP3 and VP4. The sequence of VP1, VP2, VP3 and VP4 allowed the construction of the genomic map of both IPNV and IBDV. See Dobos, P., The molecular biology of IPNV, Ann. Rev. Fish Diseases, 5:25-54 (1995).
The IPNV viral genome is contained within a non-enveloped icosahedral capsid that is approximately 60 mn in diameter. The larger “A” segment has a molecular weight of 2.5×106 Daltons (Da) and encodes at least three proteins. Their order in the genome, from the N (5′) terminus, is β(VP2) (approximately 54 kDa, major capsid protein); γ2 (NS) (an approximately 27.5 kDa nonstructural protein having proteolytic activity); and γ1 (VP3) (an approximately 31 kDa minor capsid protein. Chang et al., Can. J. Microbiol. 24:19 (1978); Huang et al., J. Virol. 60:1002 (1986). These proteins are encoded on a single mRNA within the infected cell. Mertens et al., Nature 297:243 (1982). Genome segment A of IPNV contains the a large open reading frame (ORF) encoding a 106 kDa polyprotein, which has the structure: NH2-preVP2-VP4 protease-VP3-COOH. The polyprotein is cotranslationally cleaved by a virus-encoded protease, to generate preVP2 (pVP2) and VP3. The VP2 is further cleaved during viral maturation to produce VP2. Genome segment A contains an additional small ORF, which overlaps the amino terminus of the polyprotein ORF and is in a different reading frame. This small ORF encodes a 17 kDa arginine-rich minor polypeptide, which can be detected in IPNV infected cells. The product of genome segment B is a minor internal polypeptide VP1. VP1 is the putative virion-associated RNA-dependent RNA polymerase. VP1 is present in the virions in two forms: (1) as a free polypeptide and (2) as a genome-linked protein (VPg).
The genomic maps established for IBDV are similar to the IPNV genomic map described above, which indicates that the their genomic organization is characteristic of birnaviruses in general. Thus, the unique features of birnaviruses are: (i) a genome segment A is both structurally and functionally bicistronic; (ii) production of a polyprotein that is cleaved by a virus-encoded protease; and (iii) the presence of a genome-linked protein (VPg). Furthermore, both genome segment A and genome segment B contain noncoding regions of considerable size at both of their ends. These noncoding sequences may be important for polymerase recognition, translation initiation and possibly genome packing. In addition, segment A from IPNV contains inverted terminal repeats of 14 nucleotides, which are similar to those reported for segment A of IBDV. See,Encyclop. Vir., Ed. R. G. Webster and A. Granosf, Academic Press (1995) at 143-149.
A few antiviral compounds used to treat human viruses have been tested for their ability to block IPNV infection in vitro. They are: virazole (Savan et al., J. Fish Dis. 3:437(1980); ribavirin, pyrazofurin and EICAR (5-ethynyl-1-β-ribofuranosylimidazole-carboxamide) (Migus et al., J. Gen. Virol. 47:47 (1980); Jashés et al., Antiviral Res. 29:309 (1996)). Although ribavirin was shown to inhibit IPNV replication in vitro, it had no efficacy in vivo. Migus et al., J. Gen Virol. 47:47 (1980). When EICAR was given to rainbow trout and Coho salmon fry on the first day after the fry were experimentally infected with IPNV, fewer deaths occurred among the infected fry. Moya et al., Antiviral Res. 48: 125 (2000).
In vivo assays establish the ability of the candidate compound to inhibit viral replication in vivo and/or improve morbidity and mortality. This was shown for ribavirin, which efficiently inhibits IPNV replication in vitro, yet not in vivo. Migus et al., supra (1980); Savan et al., J. Fish. Dis. 3: 437 (1980).
In order to decrease the prevalence of disease and increase the yields of farmed fish, a considerable number of antibiotics and chemicals are used to treat the water during fish farming. Examples of such antibiotics and chemicals are listed in Table 2.
TABLE 2Chemicals Administered in Fish Farming(by English or Spanish name)Acetic acidGriscofulvinAcriflavineHydroxyl methyl pyridineBacitracinIodosphoresphorus 1.7%LimeactivitySodium cyanateSodium hypochorite 130%ChioramphenicolIsoniacideChiorotetracyclineKanamicineSodium ChlorideLevamisoleDibromideNalidixic aciddichiormethyldimethylNeomycinphosphateOrganophosphatesPotassium DichromatcOxitetracycline(Potassium Bichromate)Oxoline acidDi-n-butyl tin oxidePotassium PermanganateDimetridazolePraziquantelDibromideethyleneAmmonium quaternary saltdipirididileneChloride toluene sulfonicEritromicinesodiumHexaclorinebenzene isomerSulfaguanidine“Febantel” FenotiacineSulfamerazineFlumeuineTetracyclineGentamicinTrimetoprimGentian VioletVitamin CGreen malachite
Other than the destruction of infected stocks and the decontamination of hatchery facilities, there is no current treatment comprising an isoxazol compound and/or an aminoglycoside compound to combat viral infections in an aquaculture setting that is capable of increasing the survival of aquatic animals susceptible to the viral infection. There is also a need for methods and compositions comprising isoxazol compounds and/or aminoglycoside compounds that can be administered prior to, during, and for periods of time after the susceptible species is exposed to a virus. Further, there is a need for a treatment to combat a virus in an aquaculture setting that employs an isoxazol compound and/or an aminoglycoside compound that is stable in solution. Also, there is no current aquatic food composition containing a isoxazol compound that is capable of increasing the survival of aquatic animals susceptible to a viral infection before, during, and for periods of time after the susceptible species is exposed to the virus. Accordingly, there remains a need for a method of increasing the survival of aquatic animals exposed to or infected with a virus where the method includes administering an isoxazol compound and/or an aminoglycoside compound. Hence, there is a need for a method of administering an isoxazol compound and/or an aminoglycoside compound to animals, including aquatic animals, susceptible to a viral infection in order to increase their survival when exposed to or infected with a virus.
Other than the destruction of infected stocks and the decontamination of hatchery facilities, there is no current treatment to combat IPNV in an aquaculture setting that is capable of increasing the survival of aquatic animals susceptible to IPNV infection. Also, there is no current treatment to combat IBDV in manner that is capable of increasing the survival of animals susceptible to IBDV infection. There is also a need for methods and compounds that can be administered prior to, during, and for periods of time after the susceptible species are exposed to IPNV or IBDV. Further, there is no current treatment to combat IPNV in an aquaculture setting that employs a compound that is stable in solution, particularly an isoxazol compound. Also, there is no current aquatic food composition containing a compound capable of increasing the survival of aquatic animals susceptible to IPNV infection before, during, and for periods of time after the susceptible species is exposed to IPNV. Also, there is no current food composition containing a compound capable of increasing the survival of animals susceptible to IBDV infection before, during, and for periods of time after the susceptible species is exposed to IBDV. Accordingly, there is a great need for a method of increasing the survival of animals, including aquatic animals, exposed to or infected with IPNV and IBDV, despite the need for such methods. Hence, there is a need for a method of administering a compound to aquatic animals susceptible to IPNV infection that will increase their survival when exposed to or infected with IPNV. Further, there is a need for a method of administering a compound to animals susceptible to IBDV infection that will increase their survival when exposed to or infected with IBDV.